During the last ten years, there has been a great development in the technology of optical biosensors based on surface plasmon resonance (SPR) spectroscopy, so that a range of such devices have now gained an established market position (Biacore, Texas Instruments, Intersens, BioTuL). The sensor surfaces of these and other biosensors, which are responsible for the transduction of a biospecific recognition reaction of an analyte, are often provided with a functionalized polysaccharide layer or a layer of another hydrophilic polymer, which on the one hand make it possible to bind receptor molecules covalently via functional groups (B. Johnsson, S. Löf{dot over (a)}s & G. Lindquist, Anal. Biochem. 198 (1991), 268) and, on the other hand, fulfill the function of preventing non-specific adsorption of sample components (EP-B-589 867 or German Patent Application 198 17 180.3). The nature of the hydrophilic polymer layer on the surface is that of a hydrogel, so that it is swellable as well as flexible. Both properties are desirable for the function of this layer, since on and in it receptor molecules are fixed which need to be bound so flexibly that, after immobilization, they are still capable of binding analyte molecules.
Besides the use of hydrogels, it is also customary to covalently bind receptor molecules via flexible molecules—so-called spacers—directly to sensor surfaces. From binding experiments on monolayer systems, which, owing to the principle of their construction, are substantially less flexible than hydrogels, it is known that the spacer length is crucial both for the reactivity of functional groups and for the specific binding behavior (D. D. Schlereth, J. Electroanal. Chem. 425 (1997), 77; L. Bertilsson, H. J. Butt, G. Nelles, D. D. Schlereth, Biosensors & Bioelectronics 12 (1997), 839; T. Wink, S. J. van Zuilen, A. Bult, W. P. van Bennekom, Analyst 122, 43R (1997)).
Other than in such non-swellable and only moderately flexible monolayer systems, the spacer length between the hydrogel and receptors has not hitherto received attention. No influence of the chain length on the binding behavior of the ligand molecules to the receptor was suspected, since the hydrogel per se is in any event flexible. The hitherto known hydrogels on biosensor surfaces therefore have the schematic structure shown in FIG. 1. The surface of the sensor comprises a metal layer 14, to which optionally one or more interlayers 13 are applied. The receptor 11 is bound to the hydrogel 12 via a short, inflexible spacer.
The binding of receptor molecules to the hydrogel layer may be either directed or undirected. In the case of directed binding, the receptor molecule usually has only one residue, or possibly only a few (for example fewer than three) residues of the same type, which can be reacted with the optionally derivatized hydrogel. The resulting directed binding is therefore accurately defined spatially. The receptor molecules often need to be derivatized before the reaction, so that they have a suitable reactive residue. In the case of undirected binding, however, the receptor molecule has a plurality of (for example at least three) residues of the same type, which can be reacted with the optionally derivatized hydrogel. Since only one of these residues reacts with the hydrogel, the position of the binding is spatially unpredictable. As a consequence, the binding site, or the binding sites, of the receptor is in an undefined steric arrangement with respect to the hydrogel, which in turn compromises the accessibility of these binding sites for analyte molecules and can therefore influence the binding kinetics of the analyte/receptor interactions.
U.S. Pat. No. 5,395,587 uses short- and long-chained spacers for binding biotin as a receptor molecule. However, no influence of the length of the spacer is described. Furthermore, only directed binding of receptor molecules is studied, and not undirected binding.